Practical Winery
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1 · 2 · 3 · 4 · 5 · Microbes Part 1
January/February 2010
rods, mostly single or double, held together by a polysaccharide called a glia. Some of the cells resemble Oenococcus or Pediococcus, but Acetobacter is noticeably larger when compared to reference cultures of the other bacteria (for example, to freezedried Oenococcus used for cellar additions).
In wine, Acetobacter is not always visually distinct from Lactobacillus. However, Acetobacter cells are often wider than Lactobacillus, and taper at the ends. Acetobacter chains also tend to be composed of shorter cells than Lactobacillus though not always. In culture, though not in wine, Acetobacter produces strange involution forms that resemble crudely formed, lumpy snakes made out of clay. These forms distinguish Acetobacter in culture from other rod-shaped microbes.
In culture, surface film yeasts grow readily on yeast media, often producing wrinkled colonies, though a few species produce smooth colonies. While species can be distinguished by genetic sequencing, this not commonly useful to winemakers because the remedies (eliminate headspace, increase molecular SO2) do not differ. The common practice of pushing the film out the top by adding wine to make the container overflow contaminates the cellar and is not recommended.
Acetobacter grows on bacterial media and on most yeast media. Acetobacter is a normal part of red wine flora in the cellar. A large population does not necessarily mean the wine is spoiling; it means it has the potential to spoil if air is, or becomes, available.
Lactic acid bacteria
Wines not intended for malolactic fermentation (MLF) should be kept below pH 3.4, with a molecular SO2 of at least 0.6 to 0.8 ppm. Test malic acid enzymatically before MLF to obtain a starting level; if any CO2 or haziness occurs during cellaring, check microscopically for bacteria and test for a decrease in malic acid.
If residual sugar is present, certain Lactobacillus species make acetic acid from sugar, raising the VA in stuck wines, but there is no smell because they do not produce ethyl acetate. In stuck or sweet
wines, monitor VA very closely and check microscopically for Lactobacillus every day or two. Many stuck wines are attacked by “ferocious” Lactobacillus, which produce acetic acid from sugar. Also monitor VAduring MLF and afterwards, and if possible, test D-lactic acid, produced from sugar. Check population microscopically; lactobacilli do not grow well in culture, and PCR-based tests do not presently detect all species.
After malic acid is gone, citric acid can be metabolized stoichiometrically to produce 0.1 to 0.15 g/100 ml acetic acid. Biogenic amines, regulated by some European countries, may be produced by various lactic acid bacteria even after MLF is finished.
Many heterofermentative lactic acid bacteria can produce the N-heterocycle compounds of “mousiness,” a rare taint characterized by a very unpleasant aftertaste of mouse urine or rancid nuts. The compounds are only slightly volatile at wine pH so the wines exhibit a “popcorn” or “basmati rice” aroma, even if the taint is intense once it is put into the mouth and the higher pH allows the compounds to volatilize. Studies have been proposed to distinguish a "transient mousiness" following MLF and cured by SO2 addition from a permanent taint which resists curative measures.
Saccharomyces and Zygosaccharomyces
These fermentation yeasts require residual fermentable sugars. The classic minimum level for activity is 0.5 g/L measured by enzymatic tests of glucose and fructose, though this is a arbitrary concentration. Prediction of refermen-
tation also depends on glucose/ fructose ratio, so these sugars should be measured separately, not together. Zygosaccharomyces bailii is resistant to SO2, sorbate, and benzoate, giving it a formidable advantage.
Both yeasts grow on unselective yeast media. Zygosaccharomyces cells tend to be slightly more oval than Saccharomyces, but its identity is confirmed microscopically when the sexual stage of conjugation begins in culture in one to three weeks.
When preparing to conjugate, cells grow a projection called a “shmoo tip,” distinguished from a bud by the lack of a membrane between the cell and the projection. Conjugating cells join their cytoplasm and exchange genetic material, making odd shapes often resembling dumbbells or more bizarre structures.
Most Zygosaccharomyces strains grow on acetic acid agar while Saccharomyces do not. PCR-based genetic techniques can identify Zygosaccharomyces cultures before conjugation is observed. However, in some identification systems (such as API), certain Candida yeasts cross-react with Zygosaccharomyces, so all methods, including PCR primers, should be rigorously checked against a large number of wine-related Candida species to ensure specificity. Cultures identified as Zygosaccharomyces by any technique including PCR should be kept until conjugation is observed to confirm their identity.
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